Peptide and protein mapping by 252Cf-plasma desorption mass spectrometry

ANALYTICAL

BIOCHEMISTRY

171, 113-123 (1988)

Peptide and Protein Mapping by 252Cf-Plasma Desorption Mass Spectrometry ANTHONYTSARBOPOULOS,*GERALDW.BECKER,~.

JOHN L.OCCoLow~-rz,t

AND IAN JARDINE* *Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905 and tLilly Research Laboratories, Indianapolis, Indiana 46285 Received September 1, 1987 The mapping of peptide digests by using fast atom bombardment mass spectrometry for evaluating the correctness of known or expected protein sequences is a well-established strategy. A similar approach (“PD mapping”) is described which utilizes 25~f-plasma desorption mass spectrometty (PDMS). This PD mapping approach is demonstrated and evaluated by screening the DNA-deduced sequences of recombinant interleukin-2 and human growth hormone. In the PD mapping experiment, the protein is cleaved either chemically or enzymatically and the molecular weights of the peptides predicted from the proposed amino acid sequence are compared with those determined mass spectrometrically. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptides through additional steps of enzymatic or chemical modification on the nitrocellulose-bound peptides. The PD mapping method is both fast and sensitive, requiring only low nanomole amounts per map. 0 1988 Academic prss, 1~. KEY WORDS: mass spectromctry; peptides; protein sequencing.

The primary structure of peptides and proteins is now often determined by direct translation of the appropriate gene base sequence (1,2). Inaccuracies in protein sequences can result, however, either from mistakes in reading DNA-sequencing gels or from post-translational modifications of proteins. There is always a requirement, therefore, for rapid and sensitive methods to check the accuracy of DNA-derived peptide and protein sequences. Various mass spectrometric approaches are available for such rapid checking of peptide and protein sequences. The earlier developed permethylation technique (3,4) and the reductive GC/MS method (5-7) were based on protein digestion and chemical derivatization followed by mass spectrometric analysis of the resulting small peptides; however, both techniques suffer from a lack of speed and sensitivity. 113

A more recent approach, often referred to as “FAB mapping” (8,9), is based on the fast atom bombardment (FAB) ionization technique that allows the direct ionization and analysis of large polar peptides at high sensitivity without any prior chemical derivatization (10). This technique, therefore, allows a direct comparison of the molecular weights of the peptides expected from the proposed structure after specific enzymatic or chemical cleavage with those weights experimentally determined by FABMS ( 11,12). We have developed a similar strategy based on the californium-252-plasma desorption time-of-flight mass spectrometric (PDMS)’ technique (13,14). This technique ’ Abbreviations used: PDMS, plasma desorption mass spectrometry; IL-2, interleukin-2; hGH, human growth hormone; DTT, dithiothreitol; CNBr, cyanogen bromide; TFA, trifluoroacetic acid, DMSO, dimethyl sulfoxide. 0003-2697188 $3.00 Cowright Q 1988 by Academic Press, Inc. All rights of reproductionin any form reserved.

114

TSARBOFQULOS

is already a powerful method for the determination of the molecular weight of large molecules, usually by desorption from a nitrocellulose matrix (15- 17). In the “PD mapping” experiment, after the M, determination of the intact protein, the protein is cleaved either chemically or enzymatically and the M, of the resulting peptides is determined by direct PDMS analysis of the peptide mixture. The experimentally determined M,‘s are then compared with those predicted based upon the proposed amino acid sequence. Further confirmation of the sequence assignments of individual peptides can then be made by carrying out additional steps of enzymatic or chemical modification on the nitrocellulose-bound peptides. The “PD mapping” approach is demonstrated by screening the DNA-derived sequence of recombinant interleukin-2 (IL-2) and of human growth hormone (hGH). The general properties of the PD mapping procedure (sensitivity, selectivity, resolution, mass accuracy, ease, and convenience of analysis, etc.) are discussed. MATERIALS

AND

METHODS

Materials. Recombinant interleukin-2 and synthetic growth hormone releasing factor were obtained from HoffmannLaRoche, Inc. (Nutley, NJ). Recombinant human growth hormone was from Lilly Research Laboratories (Indianapolis, IN). Glucagon, dithiothreitol (DTT), iodoacetic acid, glutathione, and cyanogen bromide (CNBr) were purchased from Sigma Chemical Co. (St. Louis, MO). Ammonium bicarbonate was obtained from Aldrich Chemical Co. (Milwaukee, WI) while Tris base was purchased from Calbiochem (La Jolla, CA). Staphylococcus aureus protease V8 and L- ltosylamido-2-phenylethyl chloromethyl ketone-treated trypsin were obtained from Cooper Biomedical (Malvem, PA). Trifluoroacetic (TFA) acid was high purity obtained from Pierce Chemical Co. (Rockford, IL)

ET AL.

and water and acetone were HPLC grade purchased from Burdick and Jackson Lab Inc. (Muskegon, MI). Enzymatic digestion. Interleukin-2 was digested with trypsin and S. aureus V8 protease for 6 h at 37°C at substrate: enzyme ratios of about 50:1 (w/w) and 25:1 (w/w), respectively, in 1% ammonium bicarbonate buffer at pH 8.4. Reduced and carboxymethylated interleukin-2 (procedure described in Ref. (18)) was subjected to trypsin and S. aureus V8 protease digestion under similar conditions to those described above. Human growth hormone was digested with trypsin in 50 mM Tris acetate buffer (pH 7.5) at 37°C for 16-20 h with a substrate:enzyme ratio of 25: 1 (w/w). The lyophilized hydrolysate was separated into two fractions by HPLC on a Brownlee Aquapore RP-300 Cs column (0.45 X 25 cm, 7 p) using a linear gradient of 0.1% aqueous TFA and 0.1% TFA in acetonitrile (B) (O-70% B, 70 min) with uv detection at 2 10 nm. Glucagon was digested with trypsin in 1% ammonium bicarbonate buffer (pH 8.4) at 37°C for 4 h. The tryptic peptide fragments were separated by HPLC on an Altex Ultrasphere ODS Cl8 column (0.46 X 25 cm, 5 p) using a linear gradient of 0.1% aqueous TFA and 0.1% TFA in acetonitrile (O-60% acetonitrile) with uv detection at 2 10 nm. CNBr cleavage. Growth hormone releasing factor, interleukin-2 (IG2) and reduced and carboxymethylated IL-2 were dissolved in 70% HCOOH. An approximately 200- to 400-fold molar excess of CNBr over the methionine present was added and the sample was incubated at 25°C for 24 h. The reaction mixture was freeze-dried several times to remove excess CNBr and HCOOH. The CNBr peptide fragments were separated by reversephase HPLC under similar conditions to those described for the tryptic digest of glucagon. Sample preparation for PDMS analysis. Aluminum sample foils were covered with a thin layer of nitrocellulose by electrospraying ( 17,19). The peptide sample (1-4 pk amount

PROTEIN

MAPPING

BY

PLASMA

DESORPTION

ranging between 400 and 2 nmol) was applied on the nitrocellulosc-coated aluminum foil, as a 1: 1 (mole:mole) solution in glutathione (20,2 1) and dried by rapidly spinning the foil sample at 2000 rpm on a bench-top centrifuge fitted with a sample stage holder. For M, determination experiments the protein sample was rinsed with 0. I- 1.O ml of a 0.1% TFA solution and spin dried before its insertion into the mass spectrometer. Mass spectra. FABMS analysis of the HPLC tryptic fractions of hGH was carried out on the first stage (BIE) of a VG ZAB-3F triple sector (B,EBJ mass spectrometer (VG Analytical, Manchester, UK) equipped with an Ion Tech fast atom gun (Ion Tech, Ltd., Teddington, UK). Approximately l-2 nmol of the peptide sample mixed with 1 ~1 of glycerol/oxalic acid was deposited onto a stainless-steel probe tip and bombarded with an 8-keV xenon beam. Mass spectra were recorded at a scan rate of 10 s per decade at an accelerating voltage of 8 kV or reduced voltage (e.g., 6 or 4 kV) for higher mass ranges. Plasma desorption mass spectra were obtained on a BIO-ION Nordic (Uppsala, Sweden) B 1N- 1OK plasma desorption time-offlight mass spectrometer using an accelerating voltage of 20 kV and a flight tube length of 15 cm. The ion source of the mass spectrometer consists of a thin IO-pCi sample of californium-252 emitting simultaneously two almost collinear fission fragments at the rate of approximately 2200 events/s. One of the fission fragments is used to desorb and ionize the peptide sample adsorbed on the nitrocellulose surface, while the associated fission fragment from the same fission event is detected providing a “start” signal for the desorption event. The secondary ions desorbed from the sample target surface are accelerated and then drift in a field free region to a “stop” detector. The signals from “start” and “stop” detectors are processed by fast electronics and then enter a time-to-digital converter, which has a maximum time resolution of 1 ns/channel and can store 64 secondary ions per primary ion.

MASS

SPECTROMETRY

115

Spectra of peptides with masses of greater than 10,000 Da were acquired over a 6- to 9-h period, whereas those of smaller peptides were accumulated over a l- to 2-h period. The recorded time-of-flight spectra were converted to mass spectra using the time centroids for H+ and Na+ as calibration peaks. The PD mass spectra are dominated by single and multiple charged protonated molecules (MH+, MH$+, MH$+) as well as by cationized molecules (MNa+, MK+) (17,22). The determined molecular weights of peptides are the isotopically averaged molecular weights. All PD mass spectra shown in this paper are background substracted. Reactions on the PDMS target. After the PDMS analysis of the sample-coated nitrocellulose foil, a small volume of the reagent of interest (2-4 ~1) is applied and spread over the surface. Then the sample foil is suspended on a holder in a reactivial which contains the reagent solution. After capping the reactivial, the reaction proceeds under controlled conditions of time and temperature. The reagent is then removed by spindrying and the chemically or enzymatically modified film is analyzed by PDMS and compared with the PDMS spectrum of the unmodified compound. This procedure is similar to the one reported by Chait and Field (23). For the DTT reduction reaction of the CNBr digest of interleukin-2, the reaction solution contained 0.1 M DTT and 0.05 M ammonium bicarbonate buffer (pH 8.34). Three microliter of this solution, i.e., 600 molar excess of DTT over the peptide concentration, was applied onto the peptide-covered nitrocellulose foil and allowed to react for 10 min at 37°C. After reaction, the sample foil was removed from its holder, spindried, and analyzed by PDMS. RESULTS

AND

DISCUSSION

Since molecular weight determination is always an important starting point in the structure determination of a protein, the mo-

116

TSARBOPOULOS

Y;PTSSsTKK~*LDLE”L‘L~LDM,LND,N~~KNPKLTRM~TFK -e* w @ Cl CS FrYPKK~TELK”L~CL~EELKPLEE”~NLIQSKNFH~RPRDLISN

-($-TM

CSh-

FIG. 1. Amino acid sequence of human interleukin-2 from Escherichiu coli. C, and T, indicate CNBr and tryptic peptides, respectively, and correspond to the symbols depicted in Figs. 2 and 3. Peptides determined by PDMS analysis are circled.

lecular weights of recombinant IL-2 and hGH were first determined by PDMS analysis. The mass measurement accuracy of the molecular weights of IL-2 and hGH as determined by PDMS was better than 0.2% (2 1). In the next step of the PD mapping of IL-2, the protein was cleaved at the methionine residues with CNBr and the CNBr-digest mixture was analyzed by PDMS. Since there are four internal methionine residues in the IL-2 sequence (Fig. l), PDMS analysis should give five signals unless the two C-terminal CNBr fragments Cd and Cs are linked by the disulfide bond (between cysteines 58 and 105), in which case four signals would be observed, i.e., C, , C2, C3, and C4.5. Instead only three signals were observed, C1 , C2, and C3 (Fig. 2A), and the observed mass values agreed well with the theoretical mass values of the C-terminal homoserine lactone CNBrpeptide fragments predicted from the DNAdeduced sequence (Table 1). It should be noted that the C, signal at m/z = 2535.6 was accompanied by a signal at m/z = 2682.3 (Fig. 2A, asterisk). This additional peptide coeluted with the C, peptide during subsequent separation of the CNBr digest by reverse-phase HPLC. This peptide, which is 147 Da heavier than the predicted C1 fragment, apparently arises from incom-

ET AL. 7 12C3

en 0 -

EI

CP

8.

’

m ‘; : 0

Cl

4.

t*3 Q +- I2 f E s $

0x2

0.1 M DTT150mM 37%.

NH4HC03

(pH 8.34)

10 min

C2

4

8002000

4000 M/Z

6000

8000

10000

FIG. 2. Positive ion PD mass spectrum of the CNBr digest of interleukin-2: (A) 400-pmol sample; (B) after 10 min DTT reaction on the nitrocellulose-bound peptide sample.

plete CNBr cleavage at the initiation methionine position, due to partial oxidation of the methionine sulfur to the sulfoxide (the residue mass of methionine sulfoxide is 147 Da), which is then resistant to CNBr cleavage.

TABLE 1 THEORETICAL AND OBSERVED MH+ VALUES OF CNBr-PEPTIDES OF INTERLEUKIN-2

Peptide

Cl C2 G C G c&i-s-c5

!kquence i-23 24-39 40-46 47-104 105-133 47-133

Observed mass value 2535.6 1842.8 902.4 6653.5’ 3390.3’ -

Theoretical mass value0

Ab

2,535.9 1.842.2 902. I ($652.7 3,369X 10,019.5

-0.3 +0.6 +0.3 +0.8 +20.5 -

“MH+ values as homowine lactone (isotopically averaged mass values). b A designates the difference between the observed and theeretied mas values. c Values obtained after DTT reduction reaction of the sample on the PDMS target foil.

PROTEIN

MAPPING

BY PLASMA

DESORPTION

Further DTT reduction reaction carried out directly on the nitrocellulose-bound peptide mixture, generated signals corresponding to all five CNBr-peptide fragments anticipated from the DNA-derived sequence (Fig. 2B and Table 1). These data indicate that the disulfide-linked CNBr-peptides Cs and C5 (& or C4-S-S-C5) were adsorbed on the nitrocellulose surface but not desorbed; desorption of the individual peptides Cs and Cs occurs only after reduction of the disulfide bond. The mass accuracy of the C5 peptide is not as good as for the other peptides, presumably because the peak in the PDMS spectrum for this peptide was not as well defined as for the other peptides (Fig. 2B). To obtain further information on the sequence of IL-2, the protein was cleaved at the C-terminal side of the basic amino acid residues lysine and arginine with trypsin and the digest mixture was analyzed by PDMS. The PD mass spectrum did not give signals due to all tryptic peptides in the mixture (Fig. 3). The mass values of the majority of the observed signals could be matched with the tryptic peptides predicted from the proposed DNA-derived amino acid sequence of IL-2 as shown in Fig. 1 and Table 2. The mass values of the observed PDMS ions were

1

r0

T’2,3

500

1000

2000

3000

4000

(MI21 FIG. 3. Positive ion PD mass spectrum of the tryptic digest of interleukin-2.

117

MASS SPECTROMETRY TABLE 2

THEORETICALAND~BSERVED MH+ VALUES OFTRYPTICPEF'IIDESOFINTERLEUKIN-2 Observed Peptide TI T2 r, T2.3 Ti.3 T4 T, T6 Tl TS T9 TlO T11 TIZ

Sequence

ma.s9 value

O-8 lo-32 9-32 IO-35 9-35 36-38 39-43 44-48 50-54 55-76 17-83 84-91 98-120 121-133

910.3 2125.9 2854.3 3065.4 3193.3 640.2 686.4 2564.7 940.5 1583.5 -

’ Isotopically averaged mass values. b A designates the difference between retical mass values.

Theoretical mas.9 value’

Ab

9 10.0 2726.2 2854.4 3065.6 3193.8 389.5 639.8 685.8 561.6 2564.9 940.1 1583.9 2628.9 1513.8

+0.3 -0.3 -0.1 -0.2 -0.5 to.4 +0.6 -0.2 f0.4 -0.4 -

the observed

and

theo-

within kO.6 Da of the theoretical mass values of the tryptic peptide fragments (Table 2). The low-molecular-weight tryptic peptides T4 and T7 could not be observed probably because of their weak binding to the nitrocellulose surface. Information on the C-terminus of the protein (position 98-133) is also absent in the spectrum, probably due to nondesorption of peptide fragments T1, and T,z from the nitrocellulose-coated target. Nevertheless, the missing information on the IL-2 C-terminus was provided either by the DTT reduction reaction on the nitrocellulose-bound CNBr-digest mixture (Fig. 2B) or by PDMS analysis of the V8 digest of IL-2 and of the reduced and carboxymethylated IL-2 (A. Tsarbopoulos and I. Jardine, unpublished results). Thus the entire amino acid sequence of IL-2 as predicted from the DNA sequence was successfully mapped by combining a series of protein chemical or enzymatic manipulations with plasma desorption mass spectrometric analysis. The absence of peptide signals in the PDMS analysis of both the CNBr and the tryptic digest of IL-2, suggests either selective

118

TSARBOPOULOS

-3068.5

m-

I 3068.3

0 1200

1600

MI2

2800

3800

FIG. 4. Positive ion PD mass spectrum of the CNBr digest of growth hormone releasing factor.

adsorption to the nitrocellulose target or selective desorption of peptide fragments in the plasma desorption experiment. The results of the DTT reduction reaction on the CNBrdigest mixture of IL-2 carried out directly on the PDMS target, indicate that the selectivity patterns observed are attributed to selective desorption of peptides in the mixture. Similar selective desorption patterns have been observed in the FAB mass spectrometric analysis of peptide mixtures (24,25), and it has recently been suggested that this is related to the hydrophobicity of the individual peptides (26). That is, the more hydrophobic the peptide, the more readily it is observed in the FABMS analysis of peptide mixtures. The results of the present work suggest that the PDMS selectivity patterns are not related to the hydrophobicity of the individual peptides. In the PDMS analysis of the tryptic digest of IL-2 for example (Table 2), the signals due to the tryptic fragments T, , and T12 are not observed, even though their hydrophobicity indices (-34 and -437, respectively) indicate that they are hydrophobic peptides (26). Selective desorption patterns were also observed in the PDMS analysis of other peptide mixtures (e.g., tryptic and CNBr digests of horse heart myoglobin and cytochrome c; CNBr digest of growth hormone releasing

ET AL.

factor; tryptic digest of glucagon). In the PDMS analysis of the CNBr digest of growth hormone releasing factor, for example (Fig. 4) a signal due to residues l-27 (MH+, m/z 3068.5) is observed in preference to a signal due to residues 28-44 (MH+, m/z 1943.1) (ratio approximately 6.6: 1). In contrast, when these peptide fragments were separated by HPLC on a reverse-phase Cl8 column it was found from their HPLC uv absorption peak areas, after correcting for the relative uv absorptivities of the amino acids and amide bonds in each peptide, that the molar ratio of the N- and C-terminal peptides in the digest was only approximately 1.3: 1. When the two peptides were individually analyzed by PDMS, each gave an intense and readily detected MH+ ion. Similarly, when the tryptic digest of glucagon is analyzed by plasma desorption mass spectrometry, a signal due to the first tryptic fragment T1 is not observed (Fig. 5) even though it is readily detected by PDMS after reverse-phase HPLC separation and isolation. In fact, from HPLC analysis, and after correction for relative uv absorptivities, the glucagon tryptic fragments T1,

M/Z FIG. 5. Positive ion PD mass spectrum of the tryptic digest of glucagon. The asterisk-denoted N-terminal sequence ions of peptide T4 also appear in the PDMS spectrum of purified peptide fragment Tq. The cationized (+Na) ions disappeared after washing of the sample on the PDMS nitrocellulose target with 0.1% TFA.

PROTEIN

MAPPING

BY PLASMA

DESORPTION

TZ, T3, and T4 were present in the original digest in an approximately 4.4:4.9: 1:2.7 ratio. In contrast, the intensity ratio from PDMS is approximately 0: 10: 1:34. It is notable that in the PD mass spectrum of the isolated Tq glucagon peptide fragment, a series of N-terminal sequence ions are present. Above m/z 600 these are the ions Ah, A,, As, Ag, C;, and C;b in the nomenclature of Roepstorff and Fohlman (27). These ions were also seen in the PD mass spectrum of nanomole quantities of the glucagon digest, as indicated in Fig. 5. Such sequence ions tend not to be so obvious when lower amounts (subnanomole) of sample are used. The problem of selective desorption in PDMS analysis of peptide digest mixtures was emphasized when direct analysis of the tryptic digest of hGH was attempted. In this case at least eight peptides were absent in the PDMS analysis. Again, there is no apparent relationship between the hydrophobicity indices of the hGH tryptic peptides and their presence or absence in the PDMS spectrum. Therefore, in order to minimize the phenomenon of selective desorption, a modified approach was followed for the mapping of

FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQK

YSFLQNPQTSLCFSESIPTPSNREETGGKSNLELLRISLLL b--@------

T5 lQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGl0

TLMGRLEDGSPRTGQKKQTYSKFDTNSHNDDALLKNYGL T14

LYCFRKDMDKVETFLRIVOCRSVEGSCGF

@FIG. 6. Amino acid sequence of human growth hormone from E. co/i. T. indicates the tryptic peptides and corresponds to the symbols depicted in Figs. 7 and 8. Peptides determined by PDMS analysis are circled.

600

119

MASS SPECTROMETRY

1400

1000

1800

M/i!

FIG. 7. Positive ion PD mass spectrum of the tryptic digest of human growth hormone (HPLC fraction 1). Asterisks denote oxidation peaks (MH + 16)+ for methionine-containing peptides T2 and T,, .

this recombinant protein (hGH). The tryptic digest of hGH (Fig. 6) was separated into two fractions by reverse-phase HPLC (an early and a late eluting fraction), thus reducing the complexity of the original digest which, in turn, reduces the complexity of the resulting PD mass spectra and which appears to minimize the selective desorption problem as well. A similar HPLC fractionation approach has been successfully applied to FABMS analysis of complex peptide mixtures (11,28). In the mass spectra of the two hGH HPLC fractions, a total of 18 peaks were identified as MH+ signals of peptides in the mixture (Figs. 7 and 8). The mass values of 16 of these signals agreed well with the theoretical mass values of the tryptic peptide fragments predicted from the DNA-derived amino acid sequence of hGH (Table 3), and indicates the high degree of correctness of the deduced structure. Two nontryptic cleavages were identified which produced the peptides T’, (L-S cleavage) and T’iO (S-L cleavage). These cleavages are presumably caused by other peptidase activities present in the tryp sin preparation. Confirmation that these cleavages had occurred was obtained after HPLC isolation of individual peptides (see below).

120

TSARBOPOULOS

12’

111

a

4 5

vd m

0 600

0 1800

1000

2400

1400

3000

3600

1800

4200

M/Z

FIG. 8. Positive ion PD mass spectrum of the tryptic digest of human growth hormone (HPLC fraction II). The asterisk denotes an oxidation peak (MH + 16)+ for the methionine-containing peptide T,, . Cationized spcties are denoted with +Na (T, , Tic, Tie, and T4). The triangle (A) indicates an (M + 31)+ species for peptide fragment T9. The cationized (+Na) ions disappeared after washing of the sample on the PDMS nitrocellulose target with 0.1% TFA.

Cationization of the molecular ions is observed in the PDMS spectra of both hGH tryptic fractions, particularly in fraction II (Fig. 8). The extent of cationization can be minimized by washing the nitrocellulosebound peptide sample with 0.1% aqueous TFA, at the expense of the small peptide fragments (Mr < 1000) which are washed off the nitrocellulose-coated target. In fact, the identification of the natriated species present in the PDMS spectrum of Fig. 8 was carried out by reanalyzing the sample after washing, which caused the (M + Na)+ ions to disappear. Removal of salts before PDMS analysis is, however, preferable. Another characteristic of these PD mass spectra is the partial shift of some of the tryptic peptide fragment peaks by 16 Da due to oxidation of methionine residues by air (tryptic fragments Tz, Ti, , and Tt7 in Figs. 7 and 8) (29). This methionine oxidation can be easily controlled by minimizing the air exposure of the sam-

ET AL.

ple, after it is adsorbed onto the nitrocellulose PDMS target. However, this oxidation can be used to advantage to uniquely identify the methionine-containing peptide fragments in a protein digest mixture in a similar fashion to the DMSO transoxidation reaction used to identify methionine-containing peptides by FABMS (30). Identification of methionines (as well as tryptophans) is particularly desirable in analysis of unknown peptides because of their unique DNA codons. The (M + 31)+ ion present for pep tide TS (Fig. 8), which did not change after washing, is probably a nitrocellulose adduct ion (+HNO?) which has been described previously by others (17) and has been observed occasionally by us. The formation and nature of such ions has not been investigated. The PDMS data in Table 3 show that almost all of the amino acid sequence of hGH, except for the peptides T3, T5, T,, and Ti4 was present in the PD mass spectra of the two tryptic peptide fractions. Analysis of the same HPLC fractions of the tryptic digest mixture of hGH by FABMS analysis also provided most of the information on the hGH sequence (Table 3). Under the conditions used for the tryptic digestion of hGH it would not be expected that the disulfide bond between T6 and Ti6 would be cleaved. Thus, the peptides Ts and Ti6 are not present in the hGH digest and are, therefore, not observed in the FABMS experiment. Since they are hydrophobic peptides (in fact Ti6 has the highest hydrophobicity index of the entire group of pep tides) they would be expected to be readily seen by FABMS if present in the mixture. The disulfide-linked peptide T6-S-S-Ti6, however, is not observed in the FABMS analysis of the mixture, but is readily observed by PDMS. The relatively low intensity ions identified as Ts and Ti6 in the PDMS of fraction II (Fig. 8) must, therefore, be fragments of the T6-S-S-T16 disulfide. Such fragmentation is often observed in PDMS of disulfide-linked peptides.

PROTEIN

MAPPING

BY

PLASMA

DESORPTION

After these experiments were completed, the tryptic peptide digest of hGH was completely separated by HPLC. It was determined by FABMS and peptide sequence analysis that all of the peptides listed in Table 3 except T6 and T16 were indeed present in the original peptide digest. That is, even the peptides which did not show up by FABMS or PDMS analysis of the two HPLC fractions were clearly identified by FABMS after purification. When the isolated T6-S-S-Tr6 pep tide was analyzed by FABMS the disulfide was readily ionized and detected and fragment ions for T6 and T16 were then also ob-

Peptide Tl T1 T2

T3 T4 TS T6

T7 T8 T9

TIO T’IO

T,, -I’12 Tt3

TM Tl5 J-16

T6-S-S-T,6 T17 T;7 Tl8

AND

121

SPECTROMETRY

served. The small peptide T14, which was absent in both the PDMS and FABMS analyses, was identified and the small hydrophilic peptides T3, T5, and T7 were found in the HPLC solvent front. These last three peptides were determined in the solvent front fraction by FABMS but not enough material was available for comparative PDMS analysis. It has been previously demonstrated, however, that PDMS analysis of mixtures of small hydrophilic peptides can be readily carried out if the PDMS sample stage is prepared by the electrospray technique without using nitrocellulose (3 1).

TABLE

THEORETICAL

MASS

3

OBSERVED PDMS MH+ VALUES OF TRYFTK PEFTIDE~ OF HUMAN GROWTH HORMONE (FRACTIONS I AND II) AND COMPARISON TO FABMS ANALYSIS

Sequence l-8 l-6 9-16 17-19 20-38 39-4 1 42-64 65-70 71-77 78-94 95-115 100-l 15 116-127 128-134 135-140 141-145 146-158 159-167

42-64and 159-167 169-178 168-178 179-191

Observed mass value 931.5 688.2 980.4

d 2342.7

d 2617.9

d 845.3 2057.4 2263.2 1745.2 1362.7 774.1 694.0 1490.7 1149.2 3762.8 1254.8 1382.9 1401.7’

Theoretical mass value4 931.1 687.9 980.2 383.4 2343.6 404.4 2617.9 762.8 845.0 2056.5 2263.5 1744.9 1362.5 773.8 693.8 626.7 1490.6 1149.3 3764.2 1254.4 1382.6 1401.6’

Ab +0.4 +0.3 +0.2 -0.9 0.0 +0.3 +0.9 -0.3 +0.3 +0.2 +0.3 +0.2 +o. 1 -0.1 -1.4 +0.4 +0.3 +0.1

Present (+) or absent (-) in FABMS’ + + + e + e e + + + + + + + + -

+ +

LIIsotopically averaged mass values. b A designates the difference between the observed and theoretical mass values. ‘The mass accuracy of the FABMS determined monoisotopic ions was always better than kO.3 Da. d These peptides were not present in HPLC fractions I or II. e These peptides were later detected by FABMS in the HPLC solvent front. ‘Arg-Ser bond is hydrolyzed but the resulting two peptides are linked by a disulfide bond (‘82Cys-S-S-189Cys).

122

TSARBOPOULOS

Even though the selective desorption problem observed in the PDMS analysis of protein digest mixtures may be minimized by HPLC partial fractionation, it clearly remains a limitation of the otherwise powerful PD mapping procedure. The observed selectivity patterns are probably related to the PDMS sample preparation procedure and to the mechanism of formation and desorption of ions in the plasma desorption process. With a clearer understanding of the nature of and reasons for the selectivity of peptide desorption, it may be possible to overcome this limitation. CONCLUSIONS

The mapping of recombinant IL-2 and hGH demonstrates that the PD mapping procedure successfully allows rapid and facile confirmation of the DNA-deduced primary structures of peptides and proteins. Coverage of the peptide and protein sequences appears to be comparable with but not identical to that obtained by FABMS. The sensitivity of the method is good, requiring approximately 200 pm01 to 2 nmol of protein for M, determination and low nanomole amounts (l-5 nmol) per map, i.e., protein digestion followed by PDMS analysis of the digest mixture. A feature of the PD mapping approach is the relatively nondestructive nature of the mass spectrometric analysis which allows additional chemical and enzymatic reactions to be carried out on the nitrocellulose-bound peptides with direct and rapid analysis of the products (3 1,32). A current disadvantage of the PD mapping procedure is the use of a time-of-flight mass spectrometer with a relatively low resolution (~500) and poor capabilities for tandem mass spectrometry (33). These drawbacks should soon be overcome with the development of better resolution time-of-flight instruments (longer flight tube (16), ion reflector (34)) and especially with the recent development of the Z52Cf-plasma desorp-

ET AL.

tion/Fourier transform ion cyclotron resonance mass spectrometric technique (35,36). Finally, it is important to emphasize the ease of operation and maintenance of the PDMS system. The advantages of the PDMS system, i.e., high sensitivity, high mass range, ease of operation, and nondestructive analysis, along with the current development of better analyzers may ultimately render the PD approach as a competitive mass spectrometric procedure to FABMS for peptide and protein mapping. ACKNOWLEDGMENTS We thank Dr. D. J. Liberato of Hoffmann-LaRoche for the samples of recombinant interleukin-2 and synthetic growth hormone releasing factor. This work was supported by NIH Grants GM 32928 and RR 02682.

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